Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum
Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum
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DOI：
10.1006/plas.2000.1487
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发表时间：
2000-11-01
期刊：
影响因子：
2.6
通讯作者：
Egelhoff, TT
中科院分区：
文献类型：
--
作者：
Levi, S;Polyakov, M;Egelhoff, TT
我们已经考虑了用于盘基网柄藻的表达载体，其编码多克隆位点上游的绿色荧光蛋白（GFP）序列，用于引入感兴趣的序列。将cDNA插入到多克隆位点中导致表达带有氨基或羧基末端GFP标签的融合蛋白，其可用于在网骨藻细胞中的荧光定位研究。平行构建体在表达的蛋白质的氨基末端融合FLAG表位标签。将每个融合盒置于允许基于反式激活Ddp2的染色体外复制的G418抗性载体中或置于允许基于自主Ddp1的复制的载体中。在两种载体类型中观察到表达稳定性的明显差异。当通过荧光显微镜分析GFP表达细胞时，当表达基于Ddp2载体时观察到表达水平的显著细胞间变异性，而当Ddp1骨架用于表达时观察到表达水平的较少细胞间变异性。(C)北京大学出版社.
We have considered expression vectors for Dictyostelium discoideum which encode a green fluorescent protein (GFP) sequence upstream of a multicloning site for introduction of sequences of interest. Insertion of cDNAs into the multicloning site results in expression of fusion protein bearing an amino- or carboxyl-terminal GFP tag which can be used for fluorescent localization studies in Dictyostelium cells. A parallel construct fuses a FLAG epitope tag at the amino terminus of expressed protein. Each fusion cartridge was placed either in a G418-resistance vector allowing transactivated Ddp2-based extrachromosomal replication or in a vector allowing autonomous Ddp1-based replication. Distinct differences in expression stability were observed in the two vector types. When GFP-expressing cells were analyzed by fluorescence microscopy, significant cell-to-cell variability in expression level was observed when expression was based on the Ddp2 vector, while less cell-to-cell variation in expression level was observed when the Ddp1 backbone was used for expression. (C) 2000 Academic Press.